In plants, surface protection is an innate defensive strategy in which microbes are directly inhibited at their first point of host contact, usually at the boundary between the host and the external environment. While studies of chemical-based leaf surface protection in plants have focused on secreted secondary metabolites, e.g., glandular trichome exudates, animal studies have focused on secreted surface proteins deployed at host/pathogen interfaces such as skin or intestinal epithelia, as reported by Gallo and Huttner (1998); and Schroder (1999), both herein incorporated by reference.
Fungi and fungi-like, e.g., oomycete, pathogens are the major causes of plant disease, resulting in annual crop losses of ˜20% worldwide, forcing extensive control by synthetic fungicides. Many of these organisms reproduce via airborne spores and transiently exploit the plant leaf surface, or phylloplane, as a starting point for host ingress. Spores of the oomycete pathogen Peronospora tabacina, the causal agent of blue mold disease on several Nicotiana species, germinate on the leaf surface by forming a germination tube, and then penetrate the plant epidermal layer with an “infection peg.” For successful phylloplane germination to occur, spores must tolerate pre-formed biochemicals present on the leaf surface.
While some surface biochemicals are presumed to leach passively from the leaf interior, e.g., sugars, others are selectively biosynthesized by specialized epidermal cells for delivery to the phylloplane. Trichomes are simple or glanded epidermal appendages that occur on most plants. Glandular secreting trichomes are found on ˜30% of vascular plants, and they produce surface-accumulated exudates that usually contain hydrophobic isoprenoids and phenylpropanoids, the latter including flavonoids, phenolics, tannins, quinones, etc. In Solanaceae plants, amphipathic sugar esters are also commonly found in glandular trichome exudates. Such compounds have been associated with insect resistance in many plants, and pest resistance is often correlated with glandular trichome density. Two well-studied cases of glandular trichome-based insect resistance are found in the plant family Solanaceae. Sugar esters produced by tall glandular trichomes (TGSTs) of primitive tomato and potato species, and the diterpenoid cembratriene-ol produced by tobacco TGSTs, have been shown to inhibit aphid infestation. Antimicrobial activities of trichome exudate compounds, particularly monoterpenoids and sesquiterpenoids, have also been reported, but are less-studied than insect resistance.
In plants, the aerial surface is referred to as the phylloplane. In a region of the phylloplane referred to as the phyllosphere, a habitat is epitomized by specialized interactions between host plants and microorganisms, both pathogenic and epiphitic. Other regions of the phyllosphere may include epidermal cell wall spaces, the spaces inside guard cell and hydathode stoma, and leaf inner air space.
In Nicotiana tabacum, phylloplane structures include guard cells, hydathodes, simple trichomes, glandular secreting trichomes with their exudates, other epidermal cells, and the cuticle. Glandular secreting trichomes, guard cells and cuticular components have been studied, and their roles in pathogen and insect interactions have been reviewed. Recent attention has been given to molecular aspects of simple trichome differentiation and development in Arabidopsis and cotton, and the apparent roles of simple trichomes in microbial disease resistance, as affected by physical impedance to disease transmitting insects and water shedding, are documented in the literature. In contrast, the structure and function(s) of hydathodes are poorly understood, even though guttation, secretion of primarily, but not exclusively, water at the leaf surface, has been observed in many species.
The best studied tissue for secretion of antimicrobial components to the epidermal surface is the glandular secreting trichome. Perhaps 30% of vascular plants possess exudating glandular trichomes. Depending on the species, varying amounts of various biochemicals, often lipophylic, often terpenes, are produced, secreted, and accumulated by this specialized tissue type. A common form of glanded trichome is the peltate type, having an aggregate of one or more specialized gland tip cells attached by a stalk to aerial plant surfaces. Secreting trichome glands of this type produce and secrete exudate into the space outside gland cells, enclosed by a cuticle surrounding the gland. Exudate chemicals may escape this containment via pores referred to as striae in the cuticle, and run down the stalk onto the epidermal surface where they are thought to primarily serve the plant as anti-insect or anti-microbe defense agents, as described in Wagner, G. J., Wang, E., Shepherd, R. W. (2004) “New approaches for studying and exploiting an old protuberance, the plant trichome.” Annals of Botany 93: 3-11, herein incorporated by reference. Insects walking on the surface may disrupt the cuticle and become immobilized by viscous exudates, or poisoned. Airborne spores or moisture-facilitated motile spores or bacteria reaching the plant surface may contact trichome exudate that has been distributed on the surface. Thus, trichome exudation has been viewed as a first line of defense against pests and pathogens. Glandular trichome exudates may contain a wide variety of chemicals, many of which are terpenoids. Other exudate constituents are flavanoids, phenolics, and sugar esters. The amount of accumulated trichome exudate can vary widely with species and growth conditions. Hydathodes, like glandular secreting trichomes, are secreting structures that are positioned to deliver biochemicals to the leaf surface.
It has long been recognized that when the soil is moist and the air is cool and humid, leaves of many plants, particularly young leaves, will bear small liquid droplets at the leaf margin or distributed on the entire surface. This moisture is often mistaken for dew, but is generally thought to be guttated water, with some solute, presumed to be primarily inorganic salts. Hydathodes may be specialized single cells at leaf margins or stalked multicellular structures as found throughout the surface, often along veins and at vein junctures, on tobacco leaves. The most important feature of hydathodes that sets them apart from simple and glandular trichomes is their intimate connection to the xylem. The hydathode “gland” consists of very loose parenchyma cells located at the end of one more small veins. This tissue is called the epithem. In most hydathodes the epithem is surrounded by a layer of tight fitting cells called the sheath, which consists of cells that have cutinized, endodermis like, adjacent walls. It has been said that there is always at least one stoma, called a water pore, in hydathode sheaths. These pores are often larger than guard cell stoma and it is generally thought that in hydathodes of most plants, the pore cannot be closed, as described in Mauseth, J. D. (1988) Plant Anatomy, The Benjamin/Cummings Publishing Co., Inc., Chapter 9, pp. 141-166, herein incorporated by reference. A possible function of hydathodes in young leaves with immature and non-functioning stomata and poorly developed vascular tissues is to facilitate acquisition of an ample supply of mineral nutrients for rapid growth by removing xylem transported nutrients into the hydathode sheath cells while allowing the water to exit through the pore. Transfer cells with plasmalemma and cell wall ingrowths that are characteristic of cells engaged in massive solute membrane transport are found in hydathode sheath cells.
Much of what is known about the structure and function of hydathodes comes from the older literature and, while very important, it is largely descriptive and does not elucidate details about cell-level mechanisms of hydathode function. For example, the diversity of solutes that may be present in guttation water is not known, or how they are delivered to it, or if guttation is restricted to young leaves. Several recent studies using sensitive, cell-selective detection methods, such as promoter-GUS localization, show distinct chitinase gene expression in hydathodes, as well as several other tissues. Similarly, intense production of auxin in developing leaf hydathodes was correlated with vascular differentiation using a fusion of a highly active synthetic auxin response element with GUS. Also, using GUS fusions with arabidopsis purine transporter genes, evidence was obtained that these transporters may be involved in retrieval from vascular fluid of nucleobases and derivatives in hydathodes, presumably to prevent their loss by guttation. Using energy-dispersive X-ray analysis, it was recently shown that in tobacco plants exposed to very high Cd, hydathodes, called short trichomes, and also glandular trichomes, secrete Cd to the extent that Cd-containing crystals form on the external surfaces of these structures. Guttation fluid of barley, Hordeum vulgare, seedlings was recently shown to contain pathogenesis-related proteins, which, it was suggested, may inhibit motile bacteria entering the plant through open hydathode water pores. As in many grasses, leaf tips of barley seedlings have hydathodes with large water pores.